Mechanisms of SARS-CoV-2 Placental Transmission

For this review, we searched multiple databases, including PubMed, Google Scholar, WHO COVID-19 database, China National Knowledge Infrastructure (CNKI), American Congress of Obstetricians and Gynecologists (ACOG) indications, and Centers for Disease Control and Prevention (CDC) database. The search terms included: “COVID-19”, “SARS-CoV-2”, “Vertical transmission”, “Severe acute respiratory syndrome coronavirus”, “Pregnancy”, “Placenta”, “Neuropilin-1”, “ACE2”, “TMPRSS2”, “Macrophages”, “Placentitis”, and “Cytokine storm”. We identified the article titles containing the keywords on PubMed and Google Scholar.

All the included research were selected and assessed only if they met the following criteria: (1)

positive maternal SARS-CoV-2 infection confirmed by PCR test;

Studies excluded in this review were letters to the editor, abstracts without full text, clinical trials, observational studies, conference proceedings, and studies published before the COVID-19 pandemic.

The human placenta acts as a physical and immunological barrier that allows the semi-allogeneic fetus to develop inside the mother's uterus and prevents microorganisms from affecting the fetus (Rackaityte and Halkias, 2020). The placental barrier consists of two main layers: stem-like, mono-nucleated cytotrophoblasts and syncytiotropho-blasts, which consist of fused cytotrophoblasts (Kapila and Khalid, 2023). During syncytialization, cytotrophoblasts fuse with the overlying syncytiotrophoblasts, forming the placental surface. Cytoplasmic protrusions from cytotrophoblasts get through between syncytiotrophoblast cells, supplying them with nutrients and organelles (Baergen et al., 2022). Hence, the latter are more resistant to pathogens.

High viral load and immunocompromising pathogens can compromise the placental defense systems and enable fetal transmission. Furthermore, constant interactions between the outer placenta layer and maternal blood damage the syncytiotrophoblasts, making them prone to infections (Robbins and Bakardjiev, 2012; Megli and Coyne, 2022; Kapila and Khalid, 2023). Since the syncytiotrophoblasts lack intercellular gap junctions, pathogens can also cross the barrier through leukocytes (Celik et al., 2020).

The tightness of the placental barrier varies in different stages of pregnancy. Virions in the maternal bloodstream enter the fetus near the extravillous trophoblast's cells in decidua or where syncytiotrophoblasts contact maternal blood. The passage is easier in the early stage when cells are yet to fully fuse into syncytiotrophoblasts and before delivery when the syncytium begins to deteriorate (Robbins and Bakardjiev, 2012). The studies reporting SARS-CoV-2 infections among pregnant women reflect the histopathological changes in the placenta (Mirbeyk et al., 2021). The physiological immuno-modulation allows normal fetal development but also seems to be the leading cause of why pregnant women are most prone to infection and its severe course in the third trimester of pregnancy. Similarly, only infections in the third trimester, particularly after the 34th week of pregnancy, were associated with an increased risk of preterm birth (Mirbeyk et al., 2021; Fallach et al., 2022). The infection is also associated with increased maternal and fetal death risk, preeclampsia, low newborn body mass, and lower appearance, pulse, grimace, activity, and respiration scores (APGAR) (Pathirathna et al., 2022).

Although the increased looseness of the placental barrier may facilitate fetal infection, SARS-CoV-2 vertical transmission is rare (Garcia-Flores et al., 2022). However, even sporadic transmission poses a significant health concern due to physiological immunosuppression and insufficient transfer of the anti-SARS-CoV-2 antibodies from the mother to the fetus (Edlow et al., 2020). In Mirbeyk et al.'s (2021) study, only 5% of nasopharyngeal samples collected from newborns of SARS-CoV-2-positive mothers were positive.

Aside from the transmission route, other factors contributing to perinatal complications have been identified. For example, COVID-19 increases the risk of venous thrombosis, which Ahmed et al. (2020) recently explained concerning Virchow's triad. Its elements, arising from vascular remodeling, are commonly found in the placentas of COVID-19-positive mothers. They disturb the blood flow in the placenta, increase vascular resistance, and can induce a compensatory increase in local blood pressure that results in vascular injury. Decreased lumen area of placental arteries and artery wall thickening occur in SARS-CoV-2-positive women independent of symptoms. The overlap of vessel wall abnormalities, blood flow abnormalities, and hypercoagulable state increases the risk of thrombosis while gradually diminishing placental functions (Khalil and Granger, 2002; Xu et al., 2020).

SARS-CoV-2 damages the placenta by inducing inflammation in the villous chamber (Bouachba et al., 2021). However, the scope of pathological changes differs significantly between stillbirth and live birth placentas in SARS-CoV-2-positive women. Konstantinidou et al. (2022) found that over 75% of the maternal intervillous space in the stillbirth placentas was obliterated, while similar changes are much rarer in live-born neonates. SARS-CoV-2-infected placentas did not differ macroscopically from their healthy equivalents but showed signs of chronic lymphoplasmacytic deciduous, villous fibrosis, capillary injury, and blood extravasation fetal vessels thrombosis (Table 1). These changes are consistent with fetal vascular malperfusion (FVM) – placental lesions that indicate abnormal perfusion of the fetal villous parenchyma, usually caused by umbilical cord obstruction (Baston-Buest et al., 2011). Features characteristic of maternal vascular malperfusion (MVM), abnormal or damaged maternal vessels, and intervillous thrombus were also more common in SARS-CoV-2-infected placenta than in their healthy counterparts (Shanes et al., 2020; Smithgall et al., 2020; Boyraz et al., 2022). MVM and FVM are among the most common histological changes in COVID-infected placentas and constitute one of the most important mechanisms by which COVID-19 affects fetal development (Hosier et al., 2020; Sharps et al., 2020).

Schwarz et al. (2020) proposed criteria to standardize the molecular identification of the virus on the fetal side of the placenta. Since then, the transmission must be confirmed by detecting the viral antigens by immunohistochemistry or viral nucleic acid by RNA scope methods. SARS-CoV-2 placentitis is defined by the coexistence of histiocytic intervillositis, perivillous fibrin deposition, and trophoblast necrosis, regardless of whether the transplacental transmission was confirmed (Watkins et al., 2021). Severe cases may be associated with positive immunostaining for SARS-CoV-2 spike protein and positive reverse-transcription polymerase chain reaction test of placental tissues (Konstantinidou et al., 2022). Aside from confirming viral RNA in the placenta, its detection in the umbilical cord also suggests diagnosis (Menter et al., 2021). Given the sporadic nature of SARS-CoV-2 vertical transmission, the morphological changes in placentas from infected newborns are yet to be systematized. Furthermore, the transmission can be asymptomatic despite histopathological changes in the placenta – such as intervillous fibrinoid depositions and intervillositis (Boncompagni et al., 2022). On the other hand, SARS-CoV-2 infection during the third trimester of pregnancy correlates with high neonatal viremia and inflammation, which could manifest as neurological symptoms, such as axial hypertonia and opisthotonos (Vivanti et al., 2020). Therefore, identifying the mechanisms of SARS-CoV-2 vertical transmission is of critical importance.

The SARS-CoV-2 virus consists of structural, such as the spike, envelope, nucleocapsid, and membrane, and non-structural proteins. Their main function is to facilitate viral infection and multiplication (Seyed Hosseini et al., 2020; Rojas-Rueda and Morales-Zamora, 2021). The virus uses the receptor-binding domain of the spike S1 protein and receptor-binding motif (RBM) of the outer surface of the ACE2 and enters the host (Figure 1). ACE2 degrades angiotensin II, inhibiting the renin–angiotensin–aldosterone pathway (Glowacka et al., 2011; Samavati and Uhal, 2020). The host protease, TMPRSS, cleaves the S protein and enables viral cell entry (Glowacka et al., 2011). After entering the host cell, the virus replicates and synthesizes its structural proteins. SARS-CoV-2 infection of the placenta can be confirmed by immunostaining for spike protein of placental tissues, and its levels are high in severe cases of COVID-19. However, the replications of SARS-CoV-2 appear to be restricted in the human placenta (Takada et al., 2022). Two spots for virus binding and mutations at the RBM occur near the spots of ACE2 and determine the scope of host infection, playing an important role in the virus mutagenicity (Chlamydas et al., 2020; Samavati and Uhal, 2020).

Fig 1.

Various cells and tissues express ACE2, including syncytial trophoblasts, cytotrophoblasts, endothelial cells, decidual cells, and vascular smooth muscle of the villi (Nobrega Cruz et al., 2021). Co-expression of both ACE2 and TMPRSS2 was considered to be the main SARS-CoV-2 entry route. The placenta is a barrier against viral and bacterial infections but seems to lack efficacy in preventing COVID-19 vertical transmission (Mao et al., 2022). Within the placenta, some tissues express both ACE2 and TMPRSS2 (Karuppan et al., 2021; Senapati et al., 2021). In the early gestation stages, co-expression of ACE2 and TMPRSS2 is high, and both proteins can be found in decidual areas (Hosier et al., 2020). As the gestational duration increases, the co-expression of ACE2 and TMPRSS2 decreases, resulting in a lower risk of viral transmission (Jing et al., 2020).

ACE2 levels are higher in the placenta than in the lungs, but their levels change at various stages of pregnancy. They may be upregulated by the infection, i.e., due to local ischemia and placental malperfusion (Yu et al., 2016). Since ACE2 expression is the highest during early pregnancy, early syncytiotrophoblast is more susceptible to high viral load (Ruan et al., 2022). ACE2 is also expressed at all stages of pregnancy, constituting a possible transmission route. Interestingly, TMPRSS2 is absent on the syncytial surface, and even its stimulated expression does not allow SARS-CoV-2 entry into the term placenta (Colson et al., 2021).

The expression of ACE2 and TMPRSS2 in the breast is yet to be extensively investigated, but available reports suggest that the risk of SARS-CoV-2 transmission with breast milk is very low (Edlow et al., 2020; Garcia-Flores et al., 2022). As such, the American College of Obstetricians and Gynecologists recommendations encourage SARS-CoV-2-positive mothers to breastfeed (Centers for Disease Control and Prevention, 2023; The American College of Obstetricians and Gynecologists, 2023).

Hudak et al. (2023) reported that 2.2% of newborns of COVID-19-positive mothers tested positive for SARS-CoV-2. While this data suggests perinatal infection, vaginal upstream transmission, while theoretically possible, seems unlikely and vaginal delivery may be chosen as a delivery route (Fenizia et al., 2021). Interestingly, the SARS-CoV-2 virus has been recently found in semen; nevertheless, the likelihood of transmission during sexual intercourse appears extremely low (Gacci et al., 2021; Donders et al., 2022).

NRP-1 is a transmembrane coreceptor of the tyrosine kinase receptor for vascular endothelial growth factor and semaphorin. NRP-1 contains an N-terminal extracellular domain consisting of A (a1–a2), B (b1–b2), and C subdomains. Its b1 subdomain is complementary with the spike proteins of SARS-CoV-2, and their binding facilitates the SARS-CoV-2 cell's entry (Abebe et al., 2021). NRP-1 is vital in angiogenesis, axon conductance, and signal transduction (Naidoo et al., 2022). Its activity drives cell survival, proliferation, differentiation, and migration (Guo and Vander Kooi, 2015). NRP-1 is highly expressed in the respiratory tract, olfactory epithelium, and placental tissues at every stage of gestation (Mayi et al., 2021; Huang et al., 2022). NRP-1 potentiates SARS-CoV-2 infectivity; the furin cleaves the S1 fragment of SARS-CoV-2's S protein and binds to the NRP-1 cell at the cell surface, constituting another entry route for COVID-19 into the host cells (Daly et al., 2020; Abebe et al., 2021). Daly et al. (2020) showed that blocking this binding with antibodies restricts viral infection. Interestingly, the expression of the NRP-1 is much higher in human lung tissue, olfactory epithelium, and the placenta than the expressions of ACE2 and TMPRSS2 (Karuppan et al., 2021). NRP-1 has also been shown to be expressed by immune cells, including placental macrophages and T cells, suggesting their potential role in viral spread (Abebe et al., 2021).

Since the expression of the NRP-1 among the placental tissues is high, it may be involved in the virus’ vertical transmission. NRP-1 is mainly found in the decidual cells, intermediate trophoblast, and syncytiotrophoblast (Abebe et al., 2021). NRP-1 is also uniquely expressed on small syncytiotrophoblast extracellular vesicles (Roy et al., 2017). Furthermore, the NRP-1 presence on the placental structures, macrophages, and monocytes is constant through all gestation stages, increasing the probability of NRP-1 being the main route of SARS-CoV-2 vertical transmission (Baston-Buest et al., 2011) (Figure 1).

Macrophages constitute the core of placental inflammatory infiltration during SARS-CoV-2 infection. They can be subdivided into distinct populations, the most important being Hofbauer cells, which derive from the fetus, and the decidual macrophages of maternal origin (Chambers et al., 2021; Mezouar et al., 2021). All of them can damage the syncytiotrophoblasts and disrupt the maternal–fetal barrier integrity, facilitating viral transmission (Schwartz et al., 2020; Mao et al., 2022). Term placenta appears to have a larger fetal than maternal population of macrophages, but the proportion is likely to change throughout pregnancy (Mezouar et al., 2019). Nevertheless, maternal macrophages seem to play a key role in SARS-CoV-2 pathogenesis. After virus uptake, macrophages switch their phenotype from the immunosuppressive M2 subtype toward the inflammatory M1 subtype and start secreting proinflammatory mediators that damage placental tissue (Yao et al., 2019; Fu et al., 2020). Furthermore, they can also transport the virus directly into the fetus (Percivalle et al., 2021).

ACE2 does not appear necessary for macrophage infection by SARS-CoV-2, but the virus does not replicate in ACE2-deficient cells (Labzin et al., 2023). On the contrary, ACE2 and CD16 overexpression is associated with enhanced viral uptake, macrophage replication, and proinflammatory immune response (Sefik et al., 2022). Infected macrophages and monocytes migrate through the organisms, crossing the maternal–fetal barrier and spreading the infection (Jafarzadeh et al., 2020). The expression of NRP-1 on macrophages is an easy target for COVID-19 infection because the NRP-1 mRNA appears to be the highest in macrophages. It further implicates that macrophages play a critical role in fetal infections (Huang et al., 2022). SARS-CoV-2 can also enter macrophages by binding its spike (S) protein to toll-like receptor 4, a type I transmembrane protein, to increase ACE2 expression and facilitate viral entry (Aboudounya and Heads, 2021).

Models of ACE2-independent viral transmission have also been proposed. SARS-CoV-2-specific antibodies can bind the virus surface to the macrophage surface IgG Fc receptor. After host cell entry, viral RNA is released and can modify endosomal signaling. This mechanism is known as antibody-dependent enhancement (Karthik et al., 2020; Wang et al., 2022). Inflammation potentiates placental damage and increases macrophage transmission and virus penetrability through the placental barrier. The susceptibility to SARS-CoV-2 infection appears to be associated with impaired trophoblast differentiation, proinflammatory and regulatory immune cell activation, enhanced placental immune response, and complement overactivation (Chen et al., 2022).

During the maternal immune response to SARS-CoV-2 placentitis, CD163-positive M2-phenotype monocytes/macrophages and T cells migrate into the intervillous space, targeting mainly the syncytiotrophoblast. The infected maternal cells easily cross the placental barrier and may infect the fetus (Argueta et al., 2022). Considering the coexistence of multiple transmission routes, NRP-1 and ACE2 included, on the placenta and immune cells at every stage of pregnancy, macrophage-driven transmission seems probable (Figure 2). However, further trials are needed to verify this hypothesis.

Fig 2.

Finally, SARS-CoV-2 can infect fetal histiocytes within the placental chorionic villus, called Hofbauer cells, causing syncytiotrophoblast injury (Reyes et al., 2017; Argueta et al., 2022). In Schwartz et al.'s study (2021), SARS-CoV-2 infected Hofbauer cells in 18.2% (4/22) and syncytiotropho-blast in 95.5% (21/22) of examined placentas. The authors suggested that SARS-CoV-2 can extend beyond the trophoblast, and most SARS-CoV-2 transplacental fetal infections do not involve Hofbauer cells (Schwartz et al., 2021). However, even without invading villi or Hofbauer cells, fetal mononuclear cells and blood cells infected with SARS-CoV-2 seem to propagate placental infection and maternal–fetal transmission (Facchetti et al., 2020; Huang et al., 2020) (Table 2).

SARS-CoV-2 enters the cell by interacting with ACE2 and NRP-1. SARS-CoV-2's spike protein has two different domains; the first domain (S1) binds to the ACE2 receptors, whereas the second (S2) interacts with the cell's membrane. The TMPRSS2 protease on the cell membrane cleaves the spite protein, enabling membrane fusion and viral entry. NRP-1's b1 subdomain binds with spike proteins of SARS-CoV-2, facilitating SARS-CoV-2 cell's entry.

SARS-CoV-2 infection activates the immune response and systemic inflammation, leading to hemodynamic changes and cytokine storm driven by interleukins, INF-γ, and TNF-α release. COVID-19 also induces INF-γ and IL-12-dependent differentiation of CD4⁺ T cells to Th1 cells in the lymph nodes, which then secrete INF-γ and IL-12, activating macrophages and cytotoxic T cells. Inflammation-associated placental injury, resulting mainly from chronic histiocytic intervillositis, can manifest as syncytiotrophoblast necrosis, FVM, MVM, lymphoplasmacytic deciduous, loss of capillaries, or extravasation of erythrocytes. These placental changes impair the integrity of the placental barrier, facilitating viral transmission. In such an environment, placental macrophages infected by SARS-CoV-2 via ACE2 and NRP-1-dependent routes cross into fetal tissues more easily, contributing to SARS-CoV-2 vertical transmission.